Solubilization of bone mineral and degradation of the organic matrix of bone depends on the formation, by osteoclasts, of an acidic extracellular compartment.
Osteoclasts are multinucleated giant cells which are responsible for bone resorption and degrade both the inorganic and organic components of bone in a local area subjacent to the matrix attachment site (Blair et al., J Cell Biol., 102:1164-1172 (1986)). Dissolution of the hydroxyapatite mineral phase is dependent upon acidification of the subosteoclastic resorption lacuna, via the action of carbonic anhydrase II and a proton pump (Vaes, J. Cell Biol., 39:676-697 (1968); Baron et al., J. Cell Biol., 101: 2210-2222 (1985); and Blair and Schlesinger, in Biology and Physiology of the Osteoclast, Rifkin and Gay, eds. (CRC Press, Boca Raton), pp. 259-287 (1992)).
V-type proton pumps are multi-subunit complexes with two distinct functional domains: a peripherally-associated cytoplasmic catalytic sector that contains 70-(subunit A), 58-(subunit B), 40- and 33-kDa (subunit E) subunits (Xie and Stone, J. Biol. Chem., 263:9859-9866 (1988)), and a proton channel, which is likely composed of 116-, 39-, and 17-kDa components (Crider et al., J. Biol. Chem., 269:17379-17381 (1994)). Considerable speculation has focused on the possibility that osteoclast-specific proton pump subunits exist, particularly because a unique osteoclast mechanism might allow controlled and specific clinical intervention for bone mass disorders such as osteoporosis.
The present invention pertains to a gene encoding a novel human 116-kDa polypeptide subunit of the osteoclast proton pump (OC-116 KDa). OC-116 KDa mRNA was found at high levels in giant cells of osteoclastomas by Northern analysis but was not detected in tumor stromal cells or in other tissues including kidney, liver, skeletal muscle and brain. OC-116 KDa mRNA was localized to multinucleated giant cells within the osteoclastoma tumor by in situ hybridization. Analysis of the deduced amino acid sequence of the polypeptide indicates that it is a membrane bound protein with at least six transmembrane domains. Thus, it appears that OC-116 kDa represents a novel human 116-kDa subunit of a proton pump which is expressed in osteoclasts in a cell-specific manner. The cell-specific expression of OC-116 KDa makes it useful as a target for therapeutic intervention in diseases with increased resorption of bone or cartilage, such as osteoporosis and osteoarthritis.
Thus, the present invention relates to a gene encoding a polypeptide or protein which is a human osteoclast proton pump subunit. In a particular embodiment, the osteoclast proton pump subunit is a 116-kDa subunit. In another embodiment, the invention also relates to a gene encoding a polypeptide or protein which is an osteoclast proton pump subunit and comprising a nucleotide sequence of SEQ ID NO: 1. The invention described herein also relates to the polypeptide or protein encoded by the described genes. The invention also pertains to isolated DNA encoding a polypeptide which is an osteoclast proton pump subunit and comprising the nucleotide sequence of SEQ ID NO: 1 or its complementary sequence or DNA which hybridizes under conditions of medium to high stringency to the nucleotide sequence of SEQ ID NO: 1 or its complement. The invention further relates to isolated DNA encoding a polypeptide which is a human osteoclast proton pump subunit and which comprises the amino acid sequence of SEQ ID NO: 3 (FIG. 2).
The invention described herein also relates to a novel polypeptide or protein which is a human 116-kDa proton pump subunit. The invention further relates to a polypeptide or protein which is an osteoclast proton pump subunit and has the amino acid sequence of SEQ ID NO: 2. The invention also relates to a polypeptide or protein which is a human proton pump subunit and which comprises the amino acid sequence of SEQ ID NO: 3.
The present invention also relates to antibodies which bind a polypeptide which is a human osteoclast proton pump subunit. For instance, polyclonal and monoclonal antibodies which bind to the described polypeptides or proteins are within the scope of the invention. The invention also pertains to DNA constructs comprising DNA encoding a polypeptide which is an osteoclast proton pump subunit, as well as to host cells stably transformed or transfected with the DNA constructs of this invention.
The present invention also relates to assays for identifying agents which alter the rate of bone degradation. In particular, the agent to be tested is administered to a test subject or added to an in vitro cell culture, and the rate of bone degradation is determined and compared with the rate of bone degradation in a control subject or cell culture which has not been treated with the test agent. An increase or decrease in the rate of bone degradation in the test animal or cell culture indicates that the tested agent alters the rate of bone degradation.
The present invention also relates to methods of treating bone mass disorders characterized by an undesirably high rate of bone degradation, such as osteoporosis and osteoarthritis. In a particular embodiment, an agent which decreases the rate of bone degradation by decreasing the activity of a 116-kDa proton pump subunit (e.g., an antagonist of OC-116 KDa) is administered in a therapeutically appropriate amount to a patient who has a detrimentally increased rate of bone degradation, thereby decreasing the patient""s bone degradation rate.
The present invention also relates to methods of treating bone mass disorders characterized by an undesirably low rate of bone degradation. In a particular embodiment, an agent which increases the rate of bone degradation by increasing the activity of a 116-kDa proton pump subunit (e.g., an agonist of OC-116 KDa) is administered in a therapeutically appropriate amount to a patient who has a detrimentally decreased rate of bone degradation, thereby increasing the patient""s bone degradation rate. Alternatively, a polypeptide which is a human 116-kDa osteoclast proton pump subunit, optionally formulated with a physiologically appropriate medium, can be administered to a subject with a detrimentally decreased rate of bone degradation. The present invention also pertains to pharmaceutical compositions comprising a polypeptide which is a human 116-kDa osteoclast proton pump subunit, or an agonist or antagonist thereof.
The polypeptides and proteins of the present invention also have utility as osteoclast cell surface markers. Expression of the described polypeptides or proteins is characteristic of osteoclasts, and is unlikely to be found in extracellular fluids such as blood, since the proteins are integral membrane proteins. Thus, these proteins can be labelled, e.g., radioactively or fluorescently, and used as cell surface markers for osteoclasts.